Illumina HiSeq 4000 sequencing; RNA-Seq analysis of bone marrow cells from mice with iron-regulatory-protein deficiency
RNA-Seq analysis of bone marrow cells from mice with iron-regulatory-protein deficiency
The official nomenclature of the mouse strain used in this study is B6.Cg-Aco1tm1.1Mwh;Ireb2tm1.1Mwh;Gt(ROSA)26Sortm1(Cre/ERT)Nat/Bgaly. This mouse strain carries conditional alelles enabling  the co-ablation of both ACO1 and IREB2 (a.k.a. IRP1 and IRP2, respectively) in mutiple organs upon injection with tamoxifen. Following deep anesthesia with a mixture of Ketamine and Xylazin, mice have been exanguinated by cardiac puncture and death was further insurred with cervical dislocation. Bone marrow cells from pooled femura, tibiae and ilia were isolated in Iscove modified Dulbecco medium using a mortar and pestle, and were filtered on a 40 µm cell strainer. Cells were sorted on a FACS Aria apparatus, based on the expression of the cKIT and LY6G markers. 3 cell populations were sorted: cKIT-high,LY6G-negative cells, corresponding to progenitors; cKIT-negative,LY6G-high cells, corresponding to polymorphonuclear neutrophils (cKIT-low,LY6G-high in mutant mice); cKIT-negative,Ly6G-low, corresponding to an intermediate differentiation stage (cKIT-low,LY6G-low in mutant mice). Bone marrow cell populations were sorted from 3 control versus 3 mutant mice. After sorting, the cells were subjected to RNA isolation. Total RNA was extracted using the ARCTURUS PicoPure RNA isolation kit (Thermo Fisher Scientific) and included a in column treatment with RNAse-free DNAseI (Qiagen) to eliminate potential genomic DNA contaminants. ultra Low RNA-Seq libraries were prepared using the SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Takara Bio Europe SAS), combined with shearing on a Covaris apparatus (Covaris Ltd.) and the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs GmbH) according to the manufacturer's protocol.